The process of mRNA–tRNA translocationJoachim Frank*†‡§, Haixiao Gao†, Jayati Sengupta†, Ning Gao*, and Derek J. Taylor*

*Howard Hughes Medical Institute and †Health Research Incorporated, Wadsworth Center, Empire State Plaza, Albany, NY 12201-0509; and‡Department of Biomedical Sciences, State University of New York, Albany, NY 12222

This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected on April 25, 2006.

Contributed by Joachim Frank, September 13, 2007 (sent for review August 6, 2007)

In the elongation cycle of translation, translocation is the process thatadvances the mRNA–tRNA moiety on the ribosome, to allow the nextcodon to move into the decoding center. New results obtained bycryoelectron microscopy, interpreted in the light of x-ray structuresand kinetic data, allow us to develop a model of the molecular eventsduring translocation.

protein synthesis � ribosome � translation � EF-G � cryo-EM

Synthesis of proteins from their building blocks, the aminoacids, is a fundamental process in the cells of all living

organisms, be it animal, plant, or bacteria. The discovery that themacromolecular assembly that facilitates this process, the ribo-some, is highly conserved in all essential parts has lent additionalcredence to the idea of the unity of all life at the molecular level.

The ribosome is a very large (2.4 MDa in eubacteria) ribonucleic-protein complex composed of two distinct subunits, the smallsubunit (30S) charged with the task of decoding the geneticmessage carried by the messenger RNA (mRNA), the large subunit(50S) to the catalysis of peptide bond formation. Instrumental forthese fundamental processes is the interaction of the ribosome withtransfer RNA (tRNA), a small L-shaped molecule that embodies inits various forms the association of each amino acid with a three-base ‘‘word’’ of the genetic code, the codon. Translation is based onthe mutual recognition, by partial Watson–Crick pairing, betweenthe codon on the mRNA and the anticodon of the tRNA carry-ing the corresponding amino acid. In facilitating tRNA selection,decoding, and the stepwise formation of the polypeptide, ribosomalRNA (rRNA) acts as both a structural framework and a catalyst.

Despite the success in the elucidation of ribosomal structure byx-ray crystallography, the detailed mechanism by which translationof mRNA code into peptide proceeds is still only scantly under-stood. One of the obstacles we face is that although the process iscomplex and dynamic, x-ray crystallography represents the mole-cule in a static form—packed in a crystal, moreover, whose verystability depends on intermolecular contacts that are largely non-physiological. Of crucial importance for the understanding of themultistep translation process is the knowledge of how the ribosomeinteracts with its ligands, notably (apart from the most crucialligands mRNA and tRNAs) the various factors catalyzing initiation,elongation, termination, and recycling. To date, with the exceptionof ribosomal complexes containing eubacterial release (RF1 orRF2) (1) or recycling (RRF) (2, 3) factors, there exists no x-raystructure of a factor–ribosome complex. The crystal structures ofindividual subunits complexed with initiation (4–6) and recycling(7) factors have also been solved; however, crystallographic data ofelongation factors bound to the ribosome are currently still notavailable. Moreover, to date, despite many efforts, no atomicstructure is available for a eukaryotic ribosome.

Increasingly, within the past decade, cryo-electron microscopy(cryo-EM) has filled this gap; in fact, the very first three-dimensional images of the ribosome (8, 9) were obtained by thistechnique well before the first x-ray structure was solved. In themean time, cryo-EM has furnished quite detailed information onthe interaction of the Escherichia coli ribosome with initiationfactors (10), elongation factors (11–14), release factors (15–18), andribosome recycling factor (19, 20). Some of the corresponding

complexes have been visualized for the 80S eukaryotic ribosome(21–23).

As the initially low-resolution maps gave way to density mapsin the subnanometer range, evidence of conformational changesboth in the ribosome and its ligands was uncovered, and qual-itative descriptions were increasingly replaced by quantitativemeasurements based on fitting and docking of x-ray structures.As a result, we can begin to piece together mechanistic modelsthat explain kinetic, genetic, and other data collected over thefive decades of ribosome research in structural terms.

The Elongation Cycle, and TranslocationIn the course of protein synthesis, tRNA occupies successivelythe universally conserved A (aminoacyl), P (peptidyl), and E(exit) sites of the mRNA-programmed ribosome. The elongationcycle of translation is a repeating, three-step process catalyzed bythe ribosome and two GTPases, EF-Tu and EF-G (eEF1A andeEF2 in eukaryotes). First, in decoding, EF-Tu delivers anaminoacylated-tRNA (aa-tRNA) molecule as part of the ternarycomplex of aa-tRNA�EF-Tu�GTP to the A/T site. The orienta-tion with which tRNA enters the ribosome is not favorable forcodon–anticodon interaction, and the anticodon stem-loop ofthe tRNA needs to be strongly kinked at the A/T site to allow theanticodon to interact with mRNA (24). If the aa-tRNA iscognate—i.e., when its three-base anticodon forms complemen-tary base pairs with the codon in the messenger RNA—GTP ishydrolyzed, and the aa-tRNA, upon dissociation of EF-Tu�GDP,is accommodated in the ribosomal A site. The second step, thepeptidyl-transferase reaction, is catalyzed by the rRNA of thelarge subunit and occurs immediately following the accommo-dation of the aa-tRNA. The transfer of the nascent peptidechain, from peptidyl-tRNA in the ribosomal P site to theaccommodated aa-tRNA in the A site, results in the ‘‘pretrans-locational’’ (PRE) state of the translating ribosome, with thedeacylated tRNA in the P site and a peptidyl-tRNA in the A site.

The final step of the elongation cycle is translocation, whichbrings the ribosome into the ‘‘posttranslocational’’ (POST) state,with an empty A site, peptidyl-tRNA in the P site, and deacylatedtRNA in the E site. Translocation is catalyzed by the binding ofEF-G and subsequent GTP hydrolysis. The movement of the tRNAmolecules is concomitant with the movement of the bound mRNAchain by the length of three bases, allowing the next codon ofmRNA in the ribosomal A site to be presented for decoding.

Each step of the elongation cycle of translation, namely decoding,peptidyl-transfer, and translocation, presents an enigma on its own.Whereas the mechanism of peptidyl-transfer has received particular

Author contributions: D.J.T. designed the animation depicting the proposed mechanism oftranslocation; H.G. performed real-space refinement fitting for the two conformations ofthe ribosome; and J.F., H.G., J.S., N.G., and D.J.T. wrote the paper.

The authors declare no conflict of interest.

See accompanying Profile on page 19668.

§To whom correspondence should be addressed. E-mail: joachim@wadsworth.org.

This article contains supporting information online at www.pnas.org/cgi/content/full/0708517104/DC1.

© 2007 by The National Academy of Sciences of the USA

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attention (see ref. 25), and at least part of the tRNA selection stepof decoding has been unraveled in molecular detail (6), themolecular processes of EF-G-mediated translocation have beenleast understood until recently. Key findings include the discoveryof the tRNA hybrid states (26) and the results of kinetic experi-ments that GTP hydrolysis precedes translocation (27).

Important cryo-EM findings relevant to translocation are thevisualization of EF-G binding to the ribosome (11, 12, 14);the visualization of tRNA in the P/E hybrid state (13, 18, 23, 28);the discovery of the ratchet motion, a rotation of the small vs.large subunit upon binding of EF-G (29) or eEF2 (22) to theribosome; and, most recently, the elucidation of the role of GTPhydrolysis in eEF2-induced translocation of the eukaryotic ri-bosome (23).

Hybrid-State tRNAs and Intersubunit ‘‘Ratchet’’ MotionIt was originally proposed in the 1960s that translocation of tRNAsmight occur independently on the two ribosomal subunits duringelongation (30, 31). Later, biochemical data such as the results fromdirect chemical footprinting led to the hybrid-state tRNA model,which describes an intermediate step in translocation (26). Here,the acceptor ends of the A- and P-site tRNAs move into the P andE sites of the large subunit, while the anticodon stem-loops of thetRNAs remain in the A and P sites of the small subunit. Thisconfiguration results in occupation by the tRNAs of the A/P andP/E hybrid states. Cryo-EM reconstructions indeed revealed struc-tural evidence for a position of tRNA in the P/E hybrid state (11,13, 18, 23); however, the A/P hybrid state has thus far eludedstructural analysis using cryo-EM or x-ray crystallography.

The initial report of hybrid-state tRNAs by Moazed and Noller(26) suggested that tRNAs occupy the hybrid state immediatelyafter peptide bond formation, but structures representing the PREribosome determined by cryo-EM (13) and x-ray crystallography(32–35) showed that in the absence of elongation factors, thetRNAs occupy the classic A/A, P/P, and E/E sites exclusively.Potential explanations for this discrepancy are that the tRNAs aresampling conformations between classic and hybrid states in thePRE ribosome (36), or that the buffer used in the various studiesstabilizes one state of binding over the other (i.e., classic overhybrid) (28).

The P/E hybrid state tRNA in the cryo-EM reconstructions wasvisualized only when EF-G was bound to the ribosome. In additionto bringing the tRNA into the P/E hybrid state, the binding of thefactor also induces a new conformation of the ribosome, where thesmall subunit is in a different orientation with respect to the largesubunit, related to the normal conformation by a counterclockwiserotation (‘‘ratchet motion’’) (29, 37) [supporting information (SI)Fig. 6 and SI Movie 1]. Evidence for the existence of an intersubunitmotion first came from small-angle neutron scatting experiments,but the precise form of this motion could not be deduced (38). Itis likely that the ratchet motion of the ribosome even occurs in theabsence of ribosomal factors and that it is coincident with themovement of tRNAs from the classic to the hybrid states, whichfacilitate translocation (39, 40). Hence, the view is emerging that,as suggested by the cryo-EM data, the binding of EF-G/eEF2 in theGTP form stabilizes the ratcheted conformation, and with it, thehybrid-state tRNAs (40, 41). The ratcheted ribosome with hybrid-state tRNAs, therefore, represents a distinct structural intermedi-ate between the PRE and POST ribosomes. In solution, it is likelythat the PRE ribosome oscillates between the ratcheted andnonratcheted states, with EF-G catalyzing translocation to thePOST ribosome. The POST ribosome, with peptidyl-tRNA in theP-site, is locked in the nonratcheted state (13).

A convincing proof that the ratchet motion is instrumental fortranslocation is contained in a series of three recent studies. In one,the motion was inhibited by the placement of a crosslink betweenproteins of the two subunits facing each other in the normalconformation of the ribosome, and complete suppression of trans-

location was observed (42). Furthermore, two FRET studies, onebulk (40) and the other single-molecule,¶ have provided conclusiveevidence that the motion inferred from the comparison of cryo-EMmaps indeed occurs in a translating ribosome.

It is noteworthy, in this context, that the kinetic parameters oftRNA movement have been investigated in detail with the use offluorescent labels (43, 44). In addition to supporting the existenceof the A/P and P/E hybrid states, these data identify distincthybrid-state intermediates. The movements of the two tRNAsbound to the A and P sites of the small subunit occur separately,such that A/A tRNA and P/E-hybrid tRNA may exist simulta-neously in an intermediate ribosomal state. In the absence ofelongation factors, single-molecule FRET measurements suggestthat the tRNAs transition between the classic, A/P-P/E hybrid, andA/A-P/E intermediate states but spend the majority of time(�60%) in the classic states (43).

Factor-Free TranslocationSome of the observations discussed above clearly show that thetransition between two dramatically different conformations ofthe ribosome and, along with it, the transition between canonicaland hybrid states of the tRNA occur spontaneously, in theabsence of EF-G/eEF2. It is compelling to link the structural anddynamic evidence to the long-known observations of factor-freetranslocation.

Reports in the very early years of ribosome studies (45–47), whenvery little was known about its biochemistry and structure, indicatedthat the ribosome is capable of performing the synthesis of oligo-phenylalanine from a poly(U) template even in the absence ofelongation factors and GTP. Subsequent work by Spirin’s group(48–50) further indicated that the treatment of the ribosome withpCMB, or the removal of ribosome protein S12 (51), stimulates therate of factor-free translocation significantly. Recent work fromRachel Green’s laboratory (52) identified three target cysteineresidues, in protein S12 (C104 and C27) and S13 (C87), whosemodification by pCMB results in an elevated factor-free translo-cation rate, albeit with decreased accuracy (see Fig. 1).

Interestingly, ribosomal protein S12 is the very protein identifiedin cryo-EM maps as the pivot of the ratchet-like motion (13) andwas shown to interact with EF-G both by cross-linking studies (53,54) and cryo-EM (13). A number of subsequent cryo-EM studiesfound that other translation factors, including IF2 (10), RF3 (18,55), and RRF (20), all shown to interact with S12, induce the sametype of intersubunit movement as EF-G (Fig. 2 and SI Fig. 7). Infact, the above-mentioned C104 residue of S12 is located exactly atthe interface between the 30S subunit and these factors. Thesecryo-EM observations thus provide a link between factor-free andfactor-promoted translocation, where S12 plays an apparent regu-latory role in controlling the intersubunit motion of the ribosome(see below for a similar role of S13).

One apparent benefit of introducing protein factors into theelongation cycle, in the course of evolution, is that it helps lower theenergetic barriers of the various transition states, because factor-free translation occurs at a much lower rate (45, 48). Translocationitself has been shown to be an exergonic reaction (56, 57), but witha high free energy of activation [�G� � 96 kJ/mol for factor-freetranslocation (58, 59)]. Kinetic studies indicated that the free energyof activation is lowered by �18 kJ/mol in the presence of EF-G, andby another 10 kJ/mol with the hydrolysis of GTP, which correspondto an �1,000-fold increase in translocation rate and another 50-foldincrease, respectively (27, 60).

¶Cornish, P. V., Ermolenko, D., Noller, H. F., Ha, T., Ribosomes Meeting, June 3–8, 2007, CapeCod, MA, p. 69 (abstr.).

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Architecture of the Ribosome: A Molecule Made to RockX-ray studies show that the ribosome possesses a unique architec-ture with built-in instabilities, among them architectural featuresconducive to the ratchet motion. As we have seen, these propertiesare apparently required for a whole range of translational steps, not

just for translocation. Among the architectural features giving riseto instability are the following.

1. The ribosome is composed of two relatively loosely coupledsubunits, a fact that prompted Spirin (31) quite early on topostulate the existence of intersubunit motion as a necessarypart of the translational process.

2. The coupling is such that rotational movement around acentral core of connections accompanied by torsional stressof these connections is prevalent among the modes shown bynormal mode analysis (61, 62). Specifically, the central bridgeB2a is formed by RNA–RNA minor groove interactions (32,63, 64). According to Valle and coworkers (13), the rotationalpivot axis passes through a point between bridges B3 and B5aon h44, just above h27 of 16S rRNA (see also ref. 61).

3. The peripheral bridges (i.e., bridges located far away from therotational pivot axis) are quite flexible and involve at least oneprotein [e.g., B4, involving S15 and the 715 stem loop of 23SrRNA (65); for definition of bridge nomenclature, see refs. 9 and19]. The bridge having to absorb the largest movement, B1b, isformed by two proteins, S13 and L5, which disengage andreengage in different constellations (13). It is interesting to lookat this bridge in the two constellations, using the quasi-atomicmodels obtained by real-space refinement (Fig. 3): in the normalconfiguration of the ribosome (macrostate I), the electrostaticcharges of regions of S13 and L5 facing each other are ofopposite polarity, lending stability to the ribosome. In theratcheted configuration (macrostate II), in contrast, the elec-trostatic charges of regions facing each other are of equalpolarity, conveying instability to the proteins and a propensityfor gliding along their interface. By extending the motionfurther, we would reach a constellation where opposite polaritieswould again face each other, creating another stabilizing inter-action. The distance traveled between the ‘‘normal’’ position(macrostate I) and the postulated second antipolar juxtapositionwould be 30 Å, which might act as a yardstick defining themaximum amplitude of ‘‘allowed’’ ratchet motion.

4. There are a large number of tertiary contacts, identified byNoller (66), that are slightly outside of the range of canonicalA-minor interactions (67) and hence would allow the gliding ofadjacent strands of rRNA without locking them into a definedposition.

5. The rRNA possesses a number of kink-turns, a new elementdiscovered in the x-ray structure of the 50S subunit of Haloarculamarismortui (68). Kink-turns are elements with built-in insta-bility, allowing extensive hinge motions of some ribosomalcomponents, among these the L1 stalk, the A-site finger (69),and the GTPase-associated center (GAC). These elements andtheir biological significance are detailed in the following.

The L1 stalk, which is implicated in the transport of the tRNA,pivots around a hinge region in h76 of 23S rRNA and has beenobserved in different positions both by cryo-EM (13, 21, 28) andx-ray crystallography (64, 70). The position of this stalk in an‘‘inward’’ position, found in macrostate II, likely blocks the exit pathof tRNA. Conversely, an ‘‘outward’’ position, found in macrostateI, leaves the exit path open. Movement from the ‘‘inward’’ to the‘‘outward’’ positions of the L1 stalk likely coordinates translocationof tRNAs by allowing ejection of E-site tRNA from the ribosome(see ref. 13).

The A-site finger is a long rRNA helix (h38) reaching from the23S rRNA into the intersubunit region where it forms a contact(termed B1a) with S13 of the small subunit. In Thermus thermophi-lus, h38 possesses a kink-turn (Kt-38). In E. coli, it has a similarregion of instability. Because the tip of the A-site finger must followthe ratchet motion of the small subunit at a peripheral location, itsbuilt-in instability may serve to reduce the activation energy for theratcheting. Shortening of the helix, presumably leading to a dis-

Fig. 1. Atomic model of the 30S subunit of the pretranslocational ribosomefrom Thermus thermophilus (ref. 34; Protein Data Bank ID code 2J00). Thisintersubunit view of the 30S highlights several important features, which arecolor-coded and labeled. Ribosomal proteins S12 and S13 interact directly withthe 50S subunit and affect ratcheting efficiency of the ribosome. The mRNAchannel passes between the head and the body of the SSU (mRNA is coloredgray). Upon EF-G binding, the top of helix 44 (h44) bends from the A sitetoward the P site. Because helix 44 is directly connected to helix 28 (h28 or the‘‘neck’’), the movement in h44 could provide torsional force on h28. GTPhydrolysis by EF-G decouples the tether between the A-site tRNA bound to thehead and the decoding center (DC) in the body of the SSU. This event wouldrelease the head to rotate about its neck and relieve the torsional force in h28.The head rotation is likely to be important for movement of the mRNA andtRNA anticodon stem-loops on the SSU.

Fig. 2. Binding of EF-G stabilizes the ratcheted conformation of the ribo-some. (a) Cryo-EM reconstruction of the pretranslocational ribosome. Thelarge subunit is colored blue, the small subunit is colored yellow, and the P-sitetRNA is colored green. (b) Cryo-EM reconstruction of the EF-G�70S complex,showing EF-G in red. The large subunit of the two reconstructions (a and b)was aligned and the small subunit of each was superimposed (c). This view,from the solvent side of the small subunit, shows that binding of EF-G inducesa counterclockwise rotation of the small subunit (pink) when compared withthe small subunit of the pretranslocational ribosome (yellow).

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ruption of B1a, results in an acceleration of translocation activity,pointing to a role of the A-site finger as a control elementmaintaining the reading frame (71).

Finally, the GAC, formed by 23S rRNA h43 and h44, is f lexiblylinked to the main body of the large subunit by h42, whichcontains kink-turn 42. According to cryo-EM studies, the mo-bility of the GAC is essential for factor binding, forming part ofan ‘‘induced fit’’ mechanism during the binding of EF-Tu (24,72), EF-G (11), RF3 (18), and IF2 (10).

Hence, in conclusion, the ribosome’s architecture is intrinsicallyunstable, conducive to making the ribosome alternate between twoconformations. In the context of translocation, the first of theseconformations (macrostate I) is required for initial factor binding,to initiate the first step of the translocation process, whereas thesecond (macrostate II) is required for staging the hybrid states,triggering GTP hydrolysis, and disengaging the firm grip that holdsthe mRNA–tRNA moiety in the decoding center, thereby initiatingthe second step of the translocation process.

Observed Conformations of EF-GEF-G-catalyzed translocation requires EF-G to assume at least fourconformations: (i) the GTP-bound, ribosome-free form; (ii) theGTP-bound conformation of EF-G on the ribosome immediatelybefore GTP hydrolysis (as binding to the ribosome induces theGTPase of EF-G); (iii) the GDP-bound conformation, in whichEF-G remains bound to the ribosome; and (iv) the GDP-boundconformation after dissociation of the factor from the ribosome. Allfour conformations have been observed structurally—by cryo-EMfor the ribosome-bound conformations and by x-ray crystallographyfor the unbound conformations (SI Fig. 8). Such conformationalalterations, specifically a hinge-like motion between the N-terminaldomains (I, II, and G�) and C-terminal domains (III–V) of EF-G,undoubtedly play a role in the physiological function of the factor.

In the 1990s, the crystal structures of GDP-bound (73) andnucleotide-free (74) EF-G were solved. Both were shown to havesimilar conformations, with disordered regions in domain 3 and theswitch 1 loop. The ribosome-free structure of EF-G�GTP has beensolved in two conformations. First, the structure of a mutantEF-G�GDPNP revealed only slight differences when comparedwith the GDP-bound form (75). Noteworthy differences betweenthese structures are seen in the P-loop, the visible regions of theswitch 1 loop, the switch 2 loop, and a slight shift at the tip of domain

IV, where a conserved histidine resides. Whereas the �-phosphateof GDPNP is ordered in the structure, the switch 1 loop is not. Thiscould be due to an artifact from crystal packing or because thecrystals were seeded from mutant EF-G�GDP crystals. Recently,the x-ray structure of an EF-G homolog, EF-G-2, complexed withGTP was reported (76). This structure possesses a fully orderedswitch 1 loop and a conformation that is distinct from the GDP-bound EF-G structure; specifically, a reorganization of domainsIII–V with respect to domains I, II, and G�. This rearrange-ment results in a 25-Å shift at the tip of domain IV when comparedwith the GDP-bound EF-G, a conformation of EF-G undoubtedlyfavorable for binding to the ribosome as evidenced by the increasedaffinity for EF-G�GTP over its GDP-bound counterpart (see ref. 77and references therein).

Attempts to find a system in the bewildering variety of observedstructures for EF-G (see, for instance, ref. 78) need to account forthe fact that the factor is in different environments, facing differentconformational constraints when placed in a crystal context versusbound to the ribosome. Conformational differences relevant for thefunctioning of the factor while performing its work on the ribosomeare best inferred from structures of EF-G bound to the ribosomein an authentic state. Because of the lack of x-ray structures for sucha complex, the existing quasi-atomic models of EF-G computedfrom cryo-EM maps are currently the only source of information.A comparison of the cryo-EM reconstructions of EF-G (11–14)bound to the ribosome with the crystal structures of the factors(assuming that these are close to the solution forms) revealed thatEF-G undergoes gross conformational changes when it binds to theribosome. These changes are again described as a hinge-like jointmotion of domains III, IV, and V with respect to domains I, II, andG� (11, 12) and are coincident with the ratchet motion (29). Thehinge-like conformational change in EF-G induced by ribosomebinding is in the same direction as that of the GDP- to GTP-boundconformations but with even greater magnitude (�27 Å) (SI Fig. 8).

The conformations of ribosome-bound EF-G before and afterGTP hydrolysis are quite likely similar to those recently elucidatedfor eEF2 by cryo-EM reconstructions of 80S–eEF2 complexes (23).The cryo-EM maps of modified eEF2 bound to the 80S ribosomein the GTP-state (using GDPNP) and GDP-state (GDP plussordarin) revealed additional, smaller conformational changes ineEF2 upon GTP hydrolysis, which include a 6-Å shift of domain IVof eEF2 toward the decoding center (23). This conformationalchange is likely responsible for decoupling the decoding centerfrom the mRNA–tRNA duplex in the A site, a prerequisite fortranslocation.

The GTP Hydrolysis Mechanism in EF-G-Catalyzed TranslocationGTPases, including EF-Tu and EF-G and their eukaryotic coun-terparts, are a family of proteins characterized by a GTP-bindingdomain, which is responsible for the binding and hydrolysis of GTP.GTPases function as molecular switches in several processes ofcellular regulation by cycling between an active, GTP-bound stateand an inactive, GDP-bound state (see ref. 79). The core, or Gdomain, is composed of three conserved features in the nucleotide-binding pocket: the phosphate-binding loop (P-loop) and the switch1 and switch 2 motifs. The P-loop binds the nucleotide via the �- and�-phosphates, while the switch 1 and 2 loops coordinate the�-phosphate of GTP.

Based on the similarities in structure, particularly of the switch 1and 2 loops and their conserved ribosomal binding site near theGAC, it is likely that the mechanism of GTP hydrolysis is conservedamong the elongation factors. In fact, this mechanism may beextended to all ribosomal binding GTPases involved in initiation,elongation, and termination because of the high conservation infactor structures and the universal GAC binding site on theribosome. However, despite the availability of several crystal struc-tures of these factors bound with various nucleotides and antibiot-ics, the mechanism of GTP hydrolysis of the factors is not well

Fig. 3. The bridge B1b, formed by S13 and L5, in the two ratchet-relatedconformations of the ribosome. (a and b) Positions of proteins S13 and L5 inthe two states. (c and d) Surface charge representation of S13-L5 in the twostates. In c (normal conformation), the charges at the interface are of oppositepolarity, leading to stabilization. In d (ratcheted conformation), the chargesare of equal polarity, leading to instability.

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understood. This is due mainly to a lack of atomic models of theelongation factors bound to the ribosome. Such a model is obviouslyimportant for the understanding of the mechanism because GTPhydrolysis by elongation factors is accelerated on the ribosome bymore than seven orders of magnitude (80).

GTPase activation of the smaller G proteins is catalyzed by thebinding of a GTPase-activating protein (GAP), which inducesconformational changes in, and thus alters the activity of, the Gproteins (79). Because there is no GAP per se for the elongationfactors, the GAC of the ribosome apparently assumes this role.Recent cryo-EM reconstructions of EF-G�70S (76) andeEF2�80S (23) complexes revealed that the switch 1 loop ofEF-G/eEF2 is in fact ordered in the GTP-bound state. AfterGTP hydrolysis, the switch 1 loop of the ribosome-bound eEF2becomes disordered, similar to that of the EF-Tu�GDP�aurodoxcrystal structure (81) and the structure of 70S�EF-Tu�Phe-tRNAPhe�kirromycin complex studied by cryo-EM, which alsorepresents a post-GTPase state (24). This observation indicatesa critical role of the switch 1 loop, as well as the existence of asimilar mechanism of GTP hydrolysis in EF-Tu and EF-G/eEF2.

The sarcin–ricin loop (SRL) of the ribosome is a conservedtetraloop in the rRNA of the large subunit, also implicated in theactivation of the GTPase activity. Cleavage of the SRL by theribotoxin �-sarcin or ricin inhibits binding of elongation factors (82,83). When free in solution, the SRL binds EF-G (84). Cryo-EMshowed that the SRL is in close proximity of the switch 1 loop ofGTP-bound EF-G and eEF2 (23, 76). Chemical protection assaysrevealed that the SRL is protected by EF-G in either the GTP-bound (GDPNP) or the GDP-bound (stabilized by fusidic acid)conformation (85). A conserved feature in the cryo-EM recon-structions of the ribosome-bound GTPases, IF2 (86) and eEF2 (23),is a shift in the GTP-binding domain of the factors toward the GACof the ribosome in the transition from the GTP-bound states toGDP-bound states. This transition may alter the interactions be-tween the SRL and the switch regions of the factors, leading to GTPhydrolysis and the disordering of the switch 1 loop.

Head Rotation of the Small Subunit: A Pivotal Stepin TranslocationWhile the movement of the tRNAs on the large subunit (i.e., theformation of the hybrid-state tRNA) is facilitated by the bindingof EF-G�GTP and the concomitant ratcheting of the ribosome,the second step of translocation, the movement of the tRNAsalong with the mRNA on the small subunit by one codon, is moredifficult to explain. Evidence has accumulated that supports thenotion that GTP hydrolysis precedes translocation on the smallsubunit (44, 87). Toe-printing experiments of EF-G�70S com-plexes in the presence of either GTP or GDPNP demonstratedthat mRNA movement relative to the small subunit occurs afterGTP hydrolysis (88). Kinetic data suggest that GTP hydrolysis byEF-G leads to some kind of ‘‘unlocking’’ event that induces aconformational change of the ribosome, on such a scale that itlimits the rate of translocation (89).

An explanation of this ‘‘unlocking’’ event was recently proposedon the basis of cryo-EM reconstructions of modified eEF2 boundto the 80S ribosome (23). Using the ADP moiety as a densitymarker, these authors showed that ADP-ribosylated eEF2 (ADPR-eEF2) undergoes conformational changes that are a direct result ofGTP hydrolysis. These changes result in a 6-Å shift in the tip ofdomain IV of ADPR-eEF2, where a diphthamide residue resides ineEF2 (or a conserved histidine, in EF-G). Judged from the locationof the tip, this movement likely severs the connection between theribosomal decoding center in the body of the small subunit and themRNA–tRNA duplex in the A site, bound to the head of the smallsubunit. Once this link is severed, the head is free to rotate aroundthe neck of the small subunit, simultaneously translocating themRNA–tRNA complex.

Such a head rotation of the subunit, which is independent ofratcheting, has been inferred from the difference in head positionsobserved both in cryo-EM reconstructions of 80S ribosomes (22,23) and in x-ray structures of the 70S ribosome (64). Even earliersmall-angle neutron scattering data predicted a head rotation of thesmall subunit to provide the mechanical act of translocation (90).Genetic mutations also suggest a necessary interaction between thehead of the small subunit and the tRNAs during translocation,because mutations to a conserved adenine in the head-region of 16SrRNA (A1339 in E. coli) confer a �18-fold decrease in translationactivity (91). Not surprisingly, A1339 interacts intimately with P-sitetRNA in the PRE ribosome (32).

Analysis of the small subunit from the x-ray structures ofvacant 70S ribosomes from E. coli revealed that residues G1338to U1341 (E. coli numbering) in the head and residue A790 inthe shoulder of the small subunit form a block between the P andE sites that opens or closes as a function of head movement (64).In the x-ray structure of the 70S ribosome in the PRE state (32)and the cryo-EM structure of the 80S ribosome in that same state(92), the distance between A1340 and A790 �-carbons is �18 Åand thus would impede movement of the tRNA stem from theP to E site. Binding of eEF2 opens this distance to �26 Å in the80S ribosome (22, 23), which would accommodate the passing ofthe anticodon stem-loop of tRNA on the small subunit from theP to the E site (64). In the cryo-EM maps, however, eEF2 isbound and this block is ‘‘open,’’ but the P/E-site tRNA remainsin the hybrid state, interacting strongly with the G1338–U1341region of the head (Fig. 4). The strong interaction seen betweenthe P/E-site tRNA and the head of the small subunit contrastswith tRNAs in the classic states, where the main interactions arewith the body of the small subunit (32–34). This result suggeststhat in a translating ribosome, the head movement physicallydrags the anticodon stem-loop of P/E site tRNA toward the Esite through a connection with the G1338–U1341 region in thehead of the small subunit after ratcheting and GTP hydrolysis.At this point, the P/E hybrid tRNA is likely to be committed tothe E site through a strong interaction formed with the L1 stalkof the large subunit after ratcheting of the ribosome (13, 21, 22),an interaction that may assist the transition phase. Disruptionsof the interaction between the head region of the small subunitand the tRNAs must be induced by a combination of back-ratcheting of the small subunit and back-rotation of the head,both of which occur upon dissociation of eEF2/EF-G�GDP. Wespeculate that it is this event that allows P/E tRNA to finally passthrough the gate between the head (G1338–U1341) and body(A790) of the small subunit to fully occupy the classic E/E state.

Finally, it is likely that the binding of EF-G indirectly provides thedriving force for head rotation in the small subunit: The binding ofEF-G�GTP and ribosomal ratcheting are coupled events that areaccompanied by a bending of h44 at a point 2/3 along its length,producing a shift of the decoding center by �8 Å toward the P site(93). This shift is required to fully accommodate domain IV ofEF-G in the ribosomal A site. Directly connected with h44 is h28in 16S rRNA, which makes up the neck of the small subunit (Fig.1). It is plausible that EF-G-induced movement at the top of h44 willcause torsional stress in h28. Thus, the head of the small subunitwould be primed to rotate as soon as the rigid link between themRNA–tRNA duplex, still bound to the small subunit’s head in theA site, and the decoding center in the body of the small subunit isremoved upon GTP hydrolysis (see Fig. 1).

Synthesis of a Model for mRNA–tRNA TranslocationRecent evidence from structural, kinetic, and biochemical data,some of which has been reviewed above, point toward a two-stepmechanism of translocation. The binding of EF-G�GTP (or EF-G�GDPNP) induces a first phase of translocation by stabilizing the

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tRNA molecules in the hybrid state. This occurs because thedeacylation of the tRNA at the P site liberates the tRNA CCA, suchthat it can proceed to the E site on the 50S subunit, and allows thesmall subunit to ratchet forward while the mRNA–tRNA remainslocked with the small subunit at its P site. At the same time, thevacating of the P site on the large subunit by the P/E-tRNA createsthe precondition for the formation of the A/P hybrid state (43).Once this state is attained and stabilized by EF-G�GTP, the secondstep of translocation will start. This step is catalyzed by GTPhydrolysis on EF-G and entails the coupled movement of themRNA and the anticodon loops of the tRNAs with respect to thesmall subunit.

The recent crystal structure of EF-G-2�GTP presents a confor-mation of EF-G that is ideally tailored to interacting with the PREribosome (76). Modeling of EF-G in this conformation with thePRE ribosome suggests that the binding of domains I, II, and G� tothe large subunit of the ribosome would position the tip of domainIV precisely at the codon–anticodon binding region of A-site tRNA

(SI Fig. 8). Domain IV of EF-G, specifically the conserved histidineresidue at its tip, comes into intimate contact with the codon–anticodon moiety formed by the mRNA and A-site tRNA. Thisinteraction could stabilize the base pairing of the codon–anticodonof A-site tRNA during ratcheting of the ribosome, as suggestedearlier (22). This reasoning would in fact explain the adverse effectson translocation when the conserved histidine in domain IV ofEF-G is mutated (94). (For the eukaryotic case, the same scenariowill hold, with EF-G replaced by eEF2 and the critical histidineresidue by diphthamide.)

The movement of the decoding center in the direction ofmRNA–tRNA translocation also stabilizes the A-site tRNA in theA/P hybrid state. Importantly, placement of tRNAs in the hybridstate is more favorable for translocation to occur as compared withtRNAs in the classic states (39, 43). Therefore, by stabilizing tRNAsin the hybrid states, EF-G binding directs the translocation processto go forward, which explains the catalytic potential of EF-G evenin the absence of GTP hydrolysis (27).

Fig. 4. The P/E-site tRNA visualized in cryo-EM reconstructions. (a) A side view of the 80S�ADPR-eEF2�GDPNP complex (23), showing the CCA end of the tRNAoccupying the E site of the large subunit, while the anticodon stem-loop occupies the P site of the small subunit. The A, P, and E sites of the ribosome areindividually labeled. (b) The anticodon stem-loop of the P/E-site tRNA forms a strong interaction with the small subunit head, near the G1338-U1341 ridge, anda weaker interaction with the small subunit body, via the A790 loop of the 18S rRNA. The gap between A790 and A1340 separates the ribosomal P and E sitesof the small subunit. (c) Stereo view of the interactions between the small subunit and P/E-site tRNA described in b. The head of the small subunit in this complexis rotated, increasing the distance between A790 in the body and A1339 in the head of the small subunit from �18 Å to �26 Å. This distance would allow passageof the anticodon stem-loop from the P to the E site; however, the tRNA remains bound in the hybrid state. Passage of the tRNA anticodon stem-loop from theP to the E site of the small subunit therefore must occur during back-ratcheting and back-rotation of the head of the small subunit, once EF-G/eEF2 dissociatesfrom the ribosome. E denotes the E site of the small subunit.

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Binding of EF-G and ratcheting of the ribosome are followed byGTP hydrolysis, causing a shift of domain IV, which in turndetaches the mRNA–tRNA complex from the decoding center(23). The mRNA–tRNA complex is temporarily freed from thebody of the small subunit but likely maintains the strong interac-tions with the subunit’s head. A head rotation in the small subunitis likely responsible for translocation of the mRNA chain by onecodon but, because the tRNAs maintain their contacts with thehead throughout the rotation, they technically remain in theirhybrid states. Disruptions of these interactions must be induced bya combination of reverse-ratcheting of the small subunit andback-rotation of the head that both occur upon release of eEF2/EF-G. The final result is the POST-state ribosome with tRNAs inthe canonical P and E sites (Fig. 5).

ConclusionThe ribosome, as we have seen, has intrinsic properties facilitatingtranslocation. The most important one is an architecture that lendsa specific kind of instability to the molecule and allows the ribosometo alternate between two distinct conformations separated by asmall activation barrier. The change from one conformation to theother is along a pathway predicted as one of the highest-rankingmodes by normal mode analysis of the x-ray structure (61, 62). Thismeans, in other words, that the architecture has developed such thata minimum of energy is needed to go from the ‘‘normal’’ confor-mation to the ‘‘ratcheted’’ conformation, which is productive for theultimate events of translocation.

This kind of parsimonious design is ubiquitous in all life forms.Human locomotion is an example on the macroscopic scale:during the act of walking, the legs are used as pendula, swingingwith close to their innate resonance frequency and thus requiringa minimum of energy for sustenance of the movement. In thesame way, energy supplied by thermal bombardment is chan-neled, by virtue of the ribosome’s unique architecture, into amotion that is most productive in terms of translation.

Cryo-EM and the methods of flexible fitting by real-spacerefinement have allowed us to produce quasi-atomic models of theribosome in both states (SI Fig. 6). An analysis of these models—particularly by watching an animation in which the two modelsalternate while the ribosome rotates (SI Movie 1)—makes it clearthat the motion involves two interrelated events: (i) a conforma-tional change in the intersubunit bridges and (ii) a change in theentire rRNA framework of each of the subunits, which resemblesan elastic deformation, and accommodates the changes in thebridges.

The principal role of EF-G/eEF2, then, is in decreasing theenergy barrier between the two ribosomal conformations. Specif-ically, the role of these factors is twofold: in bringing the ribosomeinto a conformation that is consistent with the binding of the tRNAsat the hybrid sites, and in unlocking the mRNA–tRNA moiety fromthe decoding center, thereby allowing the small subunit headmovement to occur that is required for completion of translocation.

An ‘‘ur-ribosome’’ with the same basic architecture, and the sameproperties of instability, in a world in which elongation factors hadnot yet made their appearance, would already have functioned,albeit with very much decreased efficiency and accuracy. The verysmall rate of protein synthesis in these initial stages means that theevolution of the complex G-proteins (EF-G/eEF2, EF-Tu/eEF1A,IF2/eIF5B, and RF3/eRF3) tailored precisely to the ribosome’sarchitecture could only proceed at a snail’s pace. A similar line ofreasoning holds for the impact of the addition of ribosomal proteinssuch as S12 and S13, which greatly improved the accuracy offactor-free translocation. Once these helper proteins had devel-oped, however, the resultant much higher efficiency and accuracyof protein synthesis would lead to today’s overflowing richness oflife forms.

We thank M. Watters for assistance in generating figures and Y. Chenfor help in creating SI Movie 1. This work was supported by the HowardHughes Medical Institute and National Institutes of Health Grants R37GM29169, R01 GM55440, and P41 RR01219.

1. Petry S, Brodersen DE, Murphy FV, IV, Dunham CM, Selmer M, Tarry MJ,Kelley AC, Ramakrishnan V (2005) Cell 123:1255–1266.

2. Borovinskaya MA, Pai RD, Zhang W, Schuwirth BS, Holton JM, Hirokawa G,Kaji H, Kaji A, Cate JH (2007) Nat Struct Mol Biol 14:727–732.

3. Weixlbaumer A, Petry S, Dunham CM, Selmer M, Kelley AC, RamakrishnanV (2007) Nat Struct Mol Biol 14:733–737.

4. Carter AP, Clemons WM, Jr, Brodersen DE, Morgan-Warren RJ, Hartsch T,Wimberly BT, Ramakrishnan V (2001) Science 291:498–501.

5. Pioletti M, Schlunzen F, Harms J, Zarivach R, Gluhmann M, Avila H, BashanA, Bartels H, Auerbach T, Jacobi C, et al. (2001) EMBO J 20:1829–1839.

6. Ogle JM, Brodersen DE, Clemons WM, Jr, Tarry MJ, Carter AP, Ramakrish-nan V (2001) Science 292:897–902.

7. Wilson DN, Schluenzen F, Harms JM, Yoshida T, Ohkubo T, Albrecht R,Buerger J, Kobayashi Y, Fucini P (2005) EMBO J 24:251–260.

8. Frank J, Penczek P, Grassucci R, Srivastava S (1991) J Cell Biol 115:597–605.9. Frank J, Zhu J, Penczek P, Li Y, Srivastava S, Verschoor A, Radermacher M,

Grassucci R, Lata RK, Agrawal RK (1995) Nature 376:441–444.10. Allen GS, Zavialov A, Gursky R, Ehrenberg M, Frank J (2005) Cell 121:703–712.11. Agrawal RK, Penczek P, Grassucci RA, Frank J (1998) Proc Natl Acad Sci USA

95:6134–6138.12. Agrawal RK, Heagle AB, Penczek P, Grassucci RA, Frank J (1999) Nat Struct

Biol 6:643–647.13. Valle M, Zavialov A, Sengupta J, Rawat U, Ehrenberg M, Frank J (2003) Cell

114:123–134.14. Stark H, Rodnina MV, Wieden HJ, van Heel M, Wintermeyer W (2000) Cell

100:301–309.15. Rawat U, Gao H, Zavialov A, Gursky R, Ehrenberg M, Frank J (2006) J Mol

Biol 357:1144–1153.

Fig. 5. Proposed sequence of events during the translocation process. Schematic depicts the top view of the translating ribosome. EF-G interacts with thepretranslocational ribosome (a), stabilizing the A- and P-site tRNAs into A/P and P/E hybrid sites, respectively. Binding of EF-G is coincident with the ratchetingmotion of the entire small subunit (i.e., head and body ratchet together) with respect to the small subunit (b). GTP hydrolysis causes the tip of domain IV of EF-Gto sever the connection between the small subunit head–tRNA–mRNA complex and the small subunit body (c). Once the link to the body is severed, the headrotates, translocating the mRNA by one codon and the anticodon stem-loop ends of the hybrid site tRNAs to the P and E sites (d). Translocation is completedby back-rotation of the head and reverse-ratcheting of the entire small subunit as EF-G leaves the ribosome with the tRNAs in full P/P and E/E sites (e).

Frank et al. PNAS � December 11, 2007 � vol. 104 � no. 50 � 19677

BIO

PHYS

ICS

INA

UG

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LA

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Dow

nloa

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0, 2

020

16. Rawat UB, Zavialov AV, Sengupta J, Valle M, Grassucci RA, Linde J,Vestergaard B, Ehrenberg M, Frank J (2003) Nature 421:87–90.

17. Klaholz BP, Pape T, Zavialov AV, Myasnikov AG, Orlova EV, Vestergaard B,Ehrenberg M, van Heel M (2003) Nature 421:90–94.

18. Gao H, Zhou Z, Rawat U, Huang C, Bouakaz L, Wang C, Cheng Z, Liu Y,Zavialov A, Gursky R, et al. (2007) Cell 129:929–941.

19. Agrawal RK, Sharma MR, Kiel MC, Hirokawa G, Booth TM, Spahn CM,Grassucci RA, Kaji A, Frank J (2004) Proc Natl Acad Sci USA 101:8900–8905.

20. Gao N, Zavialov AV, Li W, Sengupta J, Valle M, Gursky RP, Ehrenberg M,Frank J (2005) Mol Cell 18:663–674.

21. Gomez-Lorenzo MG, Spahn CM, Agrawal RK, Grassucci RA, Penczek P,Chakraburtty K, Ballesta JP, Lavandera JL, Garcia-Bustos JF, Frank J (2000)EMBO J 19:2710–2718.

22. Spahn CM, Gomez-Lorenzo MG, Grassucci RA, Jorgensen R, Andersen GR,Beckmann R, Penczek PA, Ballesta JP, Frank J (2004) EMBO J 23:1008–1019.

23. Taylor DJ, Nilsson J, Merrill AR, Andersen GR, Nissen P, Frank J (2007)EMBO J 26:2421–2431.

24. Valle M, Zavialov A, Li W, Stagg SM, Sengupta J, Nielsen RC, Nissen P,Harvey SC, Ehrenberg M, Frank J (2003) Nat Struct Biol 10:899–906.

25. Barta A, Dorner S, Polacek N (2001) Science 291:203.26. Moazed D, Noller HF (1989) Nature 342:142–148.27. Rodnina MV, Savelsbergh A, Katunin VI, Wintermeyer W (1997) Nature

385:37–41.28. Agrawal RK, Penczek P, Grassucci RA, Burkhardt N, Nierhaus KH, Frank J

(1999) J Biol Chem 274:8723–8729.29. Frank J, Agrawal RK (2000) Nature 406:318–322.30. Bretscher MS (1968) Nature 218:675–677.31. Spirin AS (1968) Curr Mod Biol 2:115–127.32. Yusupov MM, Yusupova GZ, Baucom A, Lieberman K, Earnest TN, Cate JH,

Noller HF (2001) Science 292:883–896.33. Korostelev A, Trakhanov S, Laurberg M, Noller HF (2006) Cell 126:1065–1077.34. Selmer M, Dunham CM, Murphy FV, IV, Weixlbaumer A, Petry S, Kelley AC,

Weir JR, Ramakrishnan V (2006) Science 313:1935–1942.35. Yusupova G, Jenner L, Rees B, Moras D, Yusupov M (2006) Nature 444:391–

394.36. Sharma D, Southworth DR, Green R (2004) RNA 10:102–113.37. Frank J, Agrawal RK (2001) Cold Spring Harbor Symp Quant Biol 66:67–75.38. Serdyuk IN, Spirin AS (1986) in Structure, Function, and Genetics of Ribosomes,

eds Hardesty B, Kramer G (Springer, New York), pp 425–437.39. Dorner S, Brunelle JL, Sharma D, Green R (2006) Nat Struct Mol Biol

13:234–241.40. Ermolenko DN, Majumdar ZK, Hickerson RP, Spiegel PC, Clegg RM, Noller

HF (2007) J Mol Biol 370:530–540.41. Spiegel PC, Ermolenko DN, Noller HF (2007) RNA 13:1473–1482.42. Horan LH, Noller HF (2007) Proc Natl Acad Sci USA 104:4881–4885.43. Munro JB, Altman RB, O’Connor N, Blanchard SC (2007) Mol Cell 25:505–

517.44. Pan D, Kirillov SV, Cooperman BS (2007) Mol Cell 25:519–529.45. Pestka S (1968) J Biol Chem 243:2810–2820.46. Pestka S (1969) J Biol Chem 244:1533–1539.47. Gavrilova LP, Smolyaninov VV (1971) Mol Biol 5:710–717.48. Gavrilova LP, Spirin AS (1971) FEBS Lett 17:324–326.49. Gavrilova LP, Spirin AS (1974) FEBS Lett 39:13–16.50. Gavrilova LP, Kostiashkina OE, Koteliansky VE, Rutkevitch NM, Spirin AS

(1976) J Mol Biol 101:537–552.51. Gavrilova LP, Koteliansky VE, Spirin AS (1974) FEBS Lett 45:324–328.52. Cukras AR, Southworth DR, Brunelle JL, Culver GM, Green R (2003) Mol Cell

12:321–328.53. Girshovich AS, Bochkareva ES, Ovchinnikov YA (1981) J Mol Biol 151:229–

243.54. Nechifor R, Wilson KS (2007) J Mol Biol 368:1412–1425.55. Klaholz BP, Myasnikov AG, Van Heel M (2004) Nature 427:862–865.56. Makarov EM, Katunin VI, Odintsov VB, Semenkov Iu P, Kirillov SV (1984)

Mol Biol (Moscow) 18:1342–1347.

57. Schilling-Bartetzko S, Bartetzko A, Nierhaus KH (1992) J Biol Chem 267:4703–4712.

58. Wintermeyer W, Savelsbergh A, Semenkov YP, Katunin VI, Rodnina MV(2001) Cold Spring Harbor Symp Quant Biol 66:449–458.

59. Semenkov YP, Shapkina TG, Kirillov SV (1992) Biochimie 74:411–417.60. Katunin VI, Savelsbergh A, Rodnina MV, Wintermeyer W (2002) Biochemistry

41:12806–12812.61. Tama F, Valle M, Frank J, Brooks CL, III (2003) Proc Natl Acad Sci USA

100:9319–9323.62. Wang Y, Rader AJ, Bahar I, Jernigan RL (2004) J Struct Biol 147:302–314.63. Cate JH, Yusupov MM, Yusupova GZ, Earnest TN, Noller HF (1999) Science

285:2095–2104.64. Schuwirth BS, Borovinskaya MA, Hau CW, Zhang W, Vila-Sanjurjo A, Holton

JM, Cate JH (2005) Science 310:827–834.65. Culver GM, Cate JH, Yusupova GZ, Yusupov MM, Noller HF (1999) Science

285:2133–2136.66. Noller HF (2005) Science 309:1508–1514.67. Nissen P, Ippolito JA, Ban N, Moore PB, Steitz TA (2001) Proc Natl Acad Sci

USA 98:4899–4903.68. Klein DJ, Schmeing TM, Moore PB, Steitz TA (2001) EMBO J 20:4214–4221.69. Osswald M, Doring T, Brimacombe R (1995) Nucleic Acids Res 23:4635–4641.70. Harms J, Schluenzen F, Zarivach R, Bashan A, Gat S, Agmon I, Bartels H,

Franceschi F, Yonath A (2001) Cell 107:679–688.71. Komoda T, Sato NS, Phelps SS, Namba N, Joseph S, Suzuki T (2006) J Biol

Chem 281:32303–32309.72. Frank J, Sengupta J, Gao H, Li W, Valle M, Zavialov A, Ehrenberg M (2005)

FEBS Lett 579:959–962.73. Czworkowski J, Wang J, Steitz TA, Moore PB (1994) EMBO J 13:3661–3668.74. Aevarsson A, Brazhnikov E, Garber M, Zheltonosova J, Chirgadze Y, al-

Karadaghi S, Svensson LA, Liljas A (1994) EMBO J 13:3669–3677.75. Hansson S, Singh R, Gudkov AT, Liljas A, Logan DT (2005) FEBS Lett

579:4492–4497.76. Connell SR, Takemoto C, Wilson DN, Wang H, Murayama K, Terada T,

Shirouzu M, Rost M, Schuler M, Giesebrecht J, et al. (2007) Mol Cell25:751–764.

77. Czworkowski J, Moore PB (1997) Biochemistry 36:10327–10334.78. Liljas A (2004) Structural Aspects of Protein Synthesis (World Scientific,

Singapore).79. Vetter IR, Wittinghofer A (2001) Science 294:1299–1304.80. Mohr D, Wintermeyer W, Rodnina MV (2002) Biochemistry 41:12520–12528.81. Vogeley L, Palm GJ, Mesters JR, Hilgenfeld R (2001) J Biol Chem 276:17149–

17155.82. Hausner TP, Atmadja J, Nierhaus KH (1987) Biochimie 69:911–923.83. Wool IG, Gluck A, Endo Y (1992) Trends Biochem Sci 17:266–269.84. Munishkin A, Wool IG (1997) Proc Natl Acad Sci USA 94:12280–12284.85. Moazed D, Robertson JM, Noller HF (1988) Nature 334:362–364.86. Myasnikov AG, Marzi S, Simonetti A, Giuliodori AM, Gualerzi CO, Yusupova

G, Yusupov M, Klaholz BP (2005) Nat Struct Mol Biol 12:1145–1149.87. Wilden B, Savelsbergh A, Rodnina MV, Wintermeyer W (2006) Proc Natl Acad

Sci USA 103:13670–13675.88. Wilson KS, Nechifor R (2004) J Mol Biol 337:15–30.89. Savelsbergh A, Katunin VI, Mohr D, Peske F, Rodnina MV, Wintermeyer W

(2003) Mol Cell 11:1517–1523.90. Serdyuk I, Baranov V, Tsalkova T, Gulyamova D, Pavlov M, Spirin A, May R

(1992) Biochimie 74:299–306.91. Abdi NM, Fredrick K (2005) RNA 11:1624–1632.92. Spahn CM, Beckmann R, Eswar N, Penczek PA, Sali A, Blobel G, Frank J

(2001) Cell 107:373–386.93. VanLoock MS, Agrawal RK, Gabashvili IS, Qi L, Frank J, Harvey SC (2000)

J Mol Biol 304:507–515.94. Savelsbergh A, Matassova NB, Rodnina MV, Wintermeyer W (2000) J Mol Biol

300:951–961.

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